Adjuvant agents that radiosensitize tumor cells without unacceptable normal tissue toxicity are potentially useful in the radiation therapy of prostate cancer. A promising candidate for enhancement of prostate tumor radiotherapy is the naturally occurring estrogen metabolite, 2-methoxyestradiol (2-ME). 2-ME interferes with microtubule function and has both anti-tumor and anti-angiogenic properties. The broad objectives of this proposal are to evaluate the radiosensitizing properties of 2-ME using both in vitro and in vivo prostate cancer models, and to study the molecular mechanisms of interaction between 2-ME and radiation. The specific aims are: 1) to investigate whether 2-ME can radiosensitize androgen-sensitive and androgen-insensitive prostate cancer models in vitro and in vivo; 2) to study the involvement of anti-angiogenesis in the mechanism of radiosensitization by 2-ME in human endothelial cells and prostate cancer xenografts; and 3) to determine the roles of MAPK, CREB, and other downstream effectors in the interaction between radiation and 2-ME in prostate cancer cells and tumors. Since current therapy for advanced prostate cancer is limited by the tendency of the disease to progress from an androgen-dependent state to an androgen-independent state, results from both androgen-dependent (LNCaP) and androgen-independent (PC3, C4-2) cell lines will be compared. Clonogenic, cell death, DNA synthesis, and other assays will be performed along with tumor xenograft (subcutaneous and orthotopic) studies in nude mice. In addition, mouse normal pelvic tissue will be histologically evaluated to assess normal tissue effects. To address the initial events of 2-ME action, the interaction between microtubules and MAPK will be examined by immunofluorescence microscopy and other quantitative techniques to test whether 2-ME disrupts the known binding between MAPK and 2-tubulin. Involvement of anti-angiogenic effects will be measured by evaluating endothelial cell proliferation (in vitro) and microvessel density (in vivo). Vascular normalization and tumor oxygenation will be addressed as potential mechanisms of radiosensitization in vivo. The roles of MAPK, CREB, HIF-11, and VEGF will be studied using traditional techniques (reporter gene assays, EMSA, and Western blotting), along with genetic modulation of these effectors. Since 2-ME as a single agent has shown anti-cancer efficacy and was well-tolerated in Phase II clinical trials against prostate cancer, these pre-clinical studies will provide important information regarding the feasibility of 2-ME as an adjuvant agent in the radiotherapy of prostate cancer. Promising results from these studies may support further clinical trials of radiation plus 2-ME in human prostate cancer patients. PUBLIC HEALTH RELEVANCE: 2-methoxyestradiol (2-ME), a naturally occurring derivative of estrogen, is a promising candidate for enhancement of prostate tumor radiotherapy, based upon our recent preliminary data in animal models. Since 2-ME as a single agent has shown limited anti-cancer activity and was well-tolerated in Phase II clinical trials against prostate cancer in humans, these pre-clinical studies will provide important information regarding the feasibility of 2-ME in combination with the radiotherapy of prostate cancer. Promising results from these studies may support further clinical trials of radiation plus 2-ME in human prostate cancer patients.